Methods of diagnosing irritable bowel syndrome and other disorders caused by small intestinal bacterial overgrowth

ABSTRACT

Disclosed is a method of diagnosing irritable bowel syndrome, fibromyalgia, chronic fatigue syndrome, depression, attention deficit/hyperactivity disorder, autoimmune diseases, such as multiple sclerosis and systemic lupus erythematosus, or Crohn&#39;s disease, which involves detecting the presence of small intestinal bacterial overgrowth (SIBO) in a human subject having at least one symptom associated with a suspected diagnosis of any of those diagnostic categories. Also disclosed is a method of treating these disorders, and other disorders caused by SIBO, that involves at least partially eradicating a SIBO condition in the human subject. The method includes administration of anti-microbial or probiotic agents, or normalizing intestinal motility by employing a prokinetic agent. The method improves symptoms, including hyperalgesia related to SIBO and disorders caused by SIBO. Also disclosed is a kit for the diagnosis or treatment of irritable bowel syndrome, fibromyalgia, chronic fatigue syndrome, depression, attention deficit/hyperactivity disorder, autoimmune diseases, or Crohn&#39;s disease.

This application is a division of Ser. No. 09/374,142, which was filedAug. 11, 1999.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referenced withinparentheses. The disclosures of these publications in their entiretiesare hereby incorporated by reference in this application in order tomore fully describe the state of the art to which this inventionpertains.

1. The Field of the Invention

This invention relates to the medical arts. It relates to a method ofdiagnosing and treating irritable bowel syndrome and other disorders,such as Crohn's disease, chronic fatigue syndrome, fibromyalgia,depression, attention deficit/hyperactivity disorder, multiplesclerosis, systemic lupus erythematosus and other autoimmune diseases ina human subject.

2. Discussion of the Related Art

Irritable bowel syndrome, Crohn's disease, chronic fatigue syndrome,fibromyalgia, depression, attention deficit/hyperactivity disorder, andautoimmune diseases, e.g., multiple sclerosis and systemic lupuserythematosus, are all clinical conditions of unclear etiology.

Irritable bowel syndrome (IBS) is the most common of allgastrointestinal disorders, affecting 11-14% of adults and accountingfor more than 50% of all patients with digestive complaints. (G.Triadafilopoulos et al., Bowel dysfunction in fibromyalgia, DigestiveDis. Sci. 36(1):59-64 [1991]; W. G. Thompson, Irritable Bowel syndrome:pathogenesis and management, Lancet 341:1569-72 [1993]). It is thoughtthat only a minority of people with IBS actually seek medical treatment.Patients with IBS present with disparate symptoms, for example,abdominal pain predominantly related to defecation, alternating diarrheaand constipation, abdominal distention, gas, and excessive mucus in thestool.

A number of possible causes for IBS have been proposed, but none hasbeen fully accepted. (W. G. Thompson [1993]). These hypotheses includeda fiber-poor Western diet, intestinal motility malfunction, abnormalpain perception, abnormal psychology or behavior, or psychophysiologicalresponse to stress.

A high fiber diet increases stool bulk and shortens gut transit time.However the presence of IBS in non-Western countries, such as China andIndia, and the failure of dietary fiber supplements to treat IBS indouble-blind clinical trials are inconsistent with the “fiberhypothesis” for the causation of IBS. (W. Bi-zhen and P. Qi-Ying,Functional bowel disorders in apparently healthy Chinese people, Chin.J. Epidemiol. 9:345-49 [1988]; K. W. Heaton, Role of dietary fibre inirritable bowel syndrome. In: R. W. Read [ed.], Irritable bowelsyndrome, Grune and Stratton, London, pp. 203-22 [1985]; W. G. Thompsonet al., Functional bowel disorders and functional abdominal pain,Gastroenterol. Int. 5:75-92 [1992]).

Those experiencing chronic IBS pain are often depressed and anxious.Treatment with tricyclic antidepressants has been used to raise the painthreshold of some IBS patients. (W. G. Thompson [1993]). Abreu et al.and Rabinovich et al. taught the use of corticotropin-releasing factorantagonists to relieve stress-related symptoms, including depression andanxiety, in IBS, anorexia nervosa, and other disorders. (M. E. Abreu,Corticotropin-releasing factor antagonism compounds, U.S. Pat. No.5,063,245; A. K. Rabinovich et al., Benzoperimidine-carboxylic acids andderivatives thereof, U.S. Pat. No. 5,861,398). Becker et al. taught theuse of serotonin antagonists to treat depression and anxiety associatedwith IBS and other conditions. (D. P Becker et al., Meso-azacyclicaromatic acid amides and esters as serotonergic agents, U.S. Pat. No.5,612,366).

Those with IBS symptoms have not been shown to have a differentpsychological or psychosocial make-up from the normal population. (W. E.Whitehead et al., Symptoms of psychologic distress associated withirritable bowel syndrome: comparison of community and medical clinicsamples, Gastroenterol. 95:709-14 [1988]). But many IBS patients appearto perceive normal intestinal activity as painful. For example, IBSpatients experience pain at lower volumes of rectal distention thannormal or have a lower than normal threshold for perceiving migratingmotor complex phase III activity. (W. E. Whitehead et al., Tolerance forrectosigmoid distension in irritable bowel syndrome, Gastroenterol.98:1187-92 [1990]; J. E. Kellow et al, Enhanced perception ofphysiological intestinal motility in the irritable bowel syndrome,Gastroenterol. 101(6):1621-27 [1991]).

Bowel motility in IBS patients differs from normal controls in responseto various stimuli such as drugs, hormones, food, and emotional stress.(D. G. Wangel and D. J. Deller, Intestinal motility in man, III:mechanisms of constipation and diarrhea with particular reference to theirritable bowel, Gastroenterol. 48:69-84 [1965]; R. F. Harvey and A. E.Read, Effect of cholecystokinin on colon motility on and symptoms inpatients with irritable bowel syndrome, Lancet i:1-3 [1973]; R. M.Valori et al., Effects of different types of stress and “prokineticdrugs” on the control of the fasting motor complex in humans,Gastroenterol. 90:1890-900 [1986]).

Evans et al. and Gorard and Farthing recognized that irritable bowelsyndrome is frequently associated with disordered gastro-intestinalmotility. (P. R. Evans et al., Gastroparesis and small bowel dysmotilityin irritable bowel syndrome, Dig. Dis. Sci. 42(10):2087-93 [1997]; D A.Gorard and M. J. Farthing, Intestinal motor function in irritable bowelsyndrome, Dig. Dis. 12(2):72-84 [1994]). Treatment directed to boweldysmotility in IBS includes the use of serotonin antagonists (D. PBecker et al., Meso-azacyclic aromatic acid amides and esters asserotonergic agents, U.S. Pat. No. 5,612,366; M. Ohta et al., Method oftreatment of intestinal diseases, U.S. Pat. No. 5,547,961) andcholecystokinin antagonists (Y. Sato et al., Benzodiazepine derivatives,U.S. Pat. No. 4,970,207; H. Kitajima et al., Thienylazole compound andthienotriazolodiazepine compound, U.S. Pat. No. 5,760,032). But colonicmotility index, altered myoelectrical activity in the colon, and smallintestinal dysmotility have not proven to be reliable diagotic tools,because they are not IBS-specific. (W. G. Thompson [1993]).

Because there has been no known underlying cause for IBS, treatment ofIBS has been primarily directed to symptoms of pain, constipation ordiarrhea symptoms.

For example, administration of the polypeptide hormone relaxin, used torelax the involuntary muscles of the intestines, is a treatment taughtto relieve the pain associated with IBS. (S. K. Yue, Method of treatingmyofascial pain syndrome with relaxin, U.S. Pat. No. 5,863,552).

Borody et al. taught the use of a picosulfate-containing laxativepreparation to treat constipation in IBS, small intestinal bacterialovergrowth, and acute or chronic bacterial bowel infections. (T. J.Borody et al., Picosulfate-containing preparation for colonicevacuation, U.S. Pat. No.5,858,403). Barody also taught the use of ananti-inflammatory agent to treat IBS. (T. J. Barody, Treatment ofnon-inflammatory and non-infectious bowel disorders, U.S. Pat. No.5,519,014). In addition, constipation in IBS has been treated withamidinourea compounds. (J. Yelnosky et al., Amidinoureas for treatingirritable bowel syndrome, U.S. Pat. Nos. 4,701,457 and 4,611,011).

Kuhla et al. taught the use of triazinone compounds to relieve IBSsymptoms of constipation, diarrhea, and abdominal pain. (D. E. Kuhla etal., Triazinones for treating irritable bowel syndrome, U.S. Pat.No.4,562,188). And Kitazawa et al. taught the use of napthy- andphenyl-sulfonylalkaoic acid compounds to treat IBS symptoms. (M.Kitazawa et al., Naphthysulfonylalkanoic acid compounds andpharmaceutical compositions thereof, U.S. Pat. No. 5,177,069; M.Kitazawa et al., Phenylsulfonylalkanoic acid compounds andpharmaceutical compositions thereof, U.S. Pat. No. 5,145,869). Daytaught an IBS treatment involving the administration of an anion-bindingpolymer and a hydrophilic polymer. (C. E. Day, Method for treatment ofirritable bowel syndrome, U.S. Pat. No. 5,380,522). And Borody et al.taught the use of salicylic acid derivatives to treat IBS. (T. J. Borodyet al., Treatment of non-inflammatory and non-infectious boweldisorders, U.S. Pat. No. 5,519,014).

A probiotic approach to the treatment of IBS has also been tried. Forexample, Allen et al. described the use of a strain of Enterococcusfaecium to alleviate symptoms. (W. D. Allen et al., Probiotic containingEnterococcus faecium strain NCIMB 40371, U.S. Pat. No. 5,728,380 andProbiotic, U.S. Pat No. 5,589,168). Borody taught a method of treatingirritable bowel syndrome by at least partial removal of the existingintestinal microflora by lavage and replacement with a new bacterialcommunity introduced by fecal inoculum from a disease-screened humandonor or by a composition comprising Bacteroides and Escherichia colispecies. (T. J. Borody, Treatment of gastro-intestinal disorders with afecal composition or a composition of bacteroides and E coli, U.S. PatNo. 5,443,826).

Fibromyalgia (FM) is a syndrome of intense generalized pain andwidespread local tenderness, usually associated with morning stiffness,fatigue, and sleep disturbances. (F. Wolfe, Fibromyalgia: the clinicalsyndrome, Rheum. Dis. Cling N. Amer. 15(1):1-17 [1989]). Fibromyalgia isoften associated with IBS (34-50% of FM cases) or other gastrointestinalsymptoms, Raynaud's phenomenon, headache, subjective swelling,paresthesias, psychological abnormality or functional disability,sometimes with overlapping symptoms of coexisting arthritis, lower backand cervical disorders, and tendonitis. Fibromyalgia affects 1-5% of thepopulation and is more prevalent among women than men. (G.Triadafilopoulos et al. [1991]).

As in IBS, a diagnosis of FM correlates with a decreased pain thresholdamong FM patients compared to non-patients. (F. Wolfe et al., Aspects ofFibromyalgia in the General Population: Sex, Pain Threshold, andFibromyalgia Symptoms, J. Rheumatol. 22:151-56 [1995]). But otherconventional laboratory evaluations of FM patients are uniformly normal.(G. Triadafilopoulos et al. [1991]). The symptoms of FM patients aretypically treated with anti-inflammatory agents and low dose tricyclicantidepressants. Administration of relaxin for involuntary muscledysfunction is also a treatment taught to relieve the pain associatedwith fibromyalgia. (S. K. Yue, Method of treating myofascial painsyndrome with relaxin, U.S. Pat. No. 5,863,552). However, there has beenno known cause of FM to which diagnosis and/or treatment could bedirected.

Chronic fatigue syndrome (CFS) affects more than a half millionAmericans. (P. H. Levine, What we know about chronic fatigue syndromeand its relevance to the practicing physician, Am. J. Med.105(3A):100S-03S [1998]). Chronic fatigue syndrome is characterized by asudden onset of persistent, debilitating fatigue and energy loss thatlasts at least six months and cannot be attributed to other medical orpsychiatric conditions; symptoms include headache, cognitive andbehavioral impairment, sore throat, pain in lymph nodes and joints, andlow grade fever. (M. Terman et al., Chronic Fatigue Syndrome andSeasonal; Affective Disorder: Comorbidity, Diagnostic Overlap, andImplications for Treatment, Am. J. Med. 105(3A):115S-24S [1998]).Depression and related symptoms are also common, including sleepdisorders, anxiety, and worsening of premenstrual symptoms or othergynecological complications. (A. L. Komaroff and D. Buchwald, Symptomsand signs of chronic fatigue syndrome, Rev. Infect. Dis. 13:S8-S11[1991]; B. L. Harlow et al., Reproductive correlates of chronic fatiguesyndrome, Am. J. Med. 105(3A):94S-99S [1998]).

Other physiologic abnormalities are also associated with CFS in manypatients, including neurally-mediated hypotension, hypocortisolism, andimmunologic dysregulation. (P. H. Levine [1998]). A subgroup of CFSpatients complain of exacerbated mood state, diminished ability to workand difficulty awakening during winter months, reminiscent of seasonalaffective disorder. (M. Terman et al [1998]).

The etiology of CFS has been unknown, and the heterogeneity of CFSsymptoms has precluded the use of any particular diagnostic laboratorytest. (P. H. Levine [1998]). Symptomatic parallels have been suggestedbetween CFS and a number of other disease conditions, resulting fromviral infection, toxic exposure, orthostatic hypotension, and stress,but none of these has been shown to have a causal role in CFS. (E.g., I.R. Bell et a., Illness from low levels of environmental chemicals:relevance to chronic fatigue syndrome and fibromyalgia, Am. J. Med.105(3A):74S-82S [1998]; R. L. Bruno et al., Parallels between post-poliofatigue and chronic fatigue syndrome: a common pathophysiology?, Am. J.Med. 105(3A):66S-73S [1998]; R. Glaser and J. K. Kiecolt-Glaser,Stress-associated immune modulation: relevance to viral infections andchronic fatigue syndrome, Am. J. Med. 105(3A):35S-42S [1998]; P. C. Roweand H. Calkins, Neurally mediated hypotension and chronic fatiguesyndrome, Am. J. Med. 105(3A):15S-21S [1998]; L. A. Jason et al.,Estimating the prevalence of chronic fatigue syndrome among nurses, Am.J. Med. 105(3A):91S-93S [1998]). One study reported that there was nosupport for an etiological role in CFS of Yersinia enterocoliticainfection. (C. M. Swanink et al., Yersinia entercolitica and the chronicfatigue syndrome, J. Infect. 36(3):269-72 [1998]). Accordingly, therehas been no known cause to which diagnosis and/or treatment of CSF couldbe directed.

Consequently, the diagnosis and treatment of CFS have continued to bedirected to symptoms, rather than to an underlying treatable cause. Forexample, the use of relaxin has been described for relaxing theinvoluntary muscles and thus relieve pain associated with CFS. (S. K.Yue, Method of treating myofascial pain syndrome with relaxin, U.S. Pat.No. 5,863,552).

Attention deficit/hyperactivity disorder (ADHD) is a heterogeneousbehaviorial disorder of unknown etiology that always appears first inchildhood, affecting 3-20% of elementary school-age children, andcontinues to affect up to 3% of adults. (Reviewed in L. L. Greenhill,Diagnosing attention deficit/hyperactivity disorder in children, J.Clin. Psychiatry 59 Suppl 7:31-41 [1998)). Those affected with ADHDsymptoms typically exhibit inattentiveness and distractability (ADtype), hyperactive and impulsive behavior (HI type), or a combination ofthese, to a degree that impairs normal functioning and is often sociallydisruptive. (M. L. Wolraich et al., Examination of DSM-IV criteria forattention deficit/hyperactivity disorder in a county-wide sample, J.Dev. Behav. Pediatr. 19(3):162-68 [1998]; J. J. Hudziak et al., Latentclass and factor analysis of DSM-IV ADHD: a twin study of femaleadolescents, J. Am. Acad. Child Adolesc. Psychiatry 37(8):848-57[1998]). Often prescribed are central nervous system stimulants,tricyclic antidepressants, antihypertensives, analgesics, or antimanicdrugs, but there has been no known cause of ADHD to which diagnosisand/or treatment could be directed. (S. C. Schneider and G. Tan,Attention deficit/hyperactivity disorder. In pursuit of diagnosticaccuracy, Postgrad. Med. 101(4):231-2,235-40 [1997]; W. J. Barbaresi,Primary-care approach to the diagnosis and management of attentiondeficit/hyperactivity disorder, Mayo Clin. Proc. 71(5):463-71 [1996]).

There has also been no known cause for autoimmune diseases, includingmultiple sclerosis and systemic lupus erythematosus. Multiple sclerosis(MS) is a neurologic disease that primarily strikes teens and youngadults under 35 years. Affecting 350,000 Americans, MS is the mostfrequent cause of neurologic disability except for traumatic injuries;MS affects twice as many females compared to males. (S. L. Hauser,Multiple Sclerosis and other demyelinating diseases In: Harrison'sPrinciples of Internal Medicine, 13th ed., K. J. Isselbacher et al.(eds.), McGraw-Hill, pp.2287-95 [1994]). The disease is characterized bychronic inflammation, scarring, and selective destruction of the myelinsheath around neural axons of the central nervous system, and is thoughtto be caused by autoimmune responses. A treatment for MS taught byWeiner et al. is related to oral administration of autoantigens to thepatient to suppress the autoimmune response by eliciting suppressorT-cells specific for myelin basic protein (MBP). There are no specificdiagnostic tests for MS; diagnosis is based on clinical recognition ofdestructive patterns of central nervous system injury that are producedby the disease. (S. L. Hauser [1994]) Nerve damage may be mediated bycytokines, especially TNF-α, which has been found to be selectivelytoxic to myelin and to oligodendrocytes in vitro. Elevated levels ofTNF-α and IL-2 were measured in MS patients. (J. L. Trotter et al.,Serum cytokine levels in chronic progressive multiple sclerosis:interleukin-2 levels parallel tumor necrosis factor-alpha levels, J.Neuroimmunol. 33(1):29-36 [1991]; H. L. Weiner et al., Treatment ofmultiple sclerosis by oral administration of autoantigens, U.S. Pat. No.5,869,054). Another treatment for MS involves the administration of avitamin D compound. (H. F. DeLuca et al., Multiple sclerosis treatment,U.S. Pat. No. 5,716,946). However, there has been no known cause of MSto which diagnosis and/or treatment could be directed.

Systemic lupus erythematosus (SLE) is an autoimmune rheumatic diseasecharacterized by deposition in tissues of autoantibodies and immunecomplexes leading to tissue injury (B. L. Kotzin, Systemic lupuserythematosus, Cell 85:303-06 [1996]). In contrast to autoimmunediseases such as MS and type 1 diabetes mellitus, SLE potentiallyinvolves multiple organ systems directly, and its clinicalmanifestations are diverse and variable. (Reviewed by B. L. Kotzin andJ. R. O'Dell, Systemic lupus erythematosus, In: Samler's ImmunologicDiseases, 5th ed., M. M. Frank et al., eds., Little Brown & Co., Boston,pp. 667-97 [1995]). For example, some patients may demonstrate primarilyskin rash and joint pain, show spontaneous remissions, and requirelittle medication. At the other end of the spectrum are patients whodemonstrate severe and progressive kidney involvement that requirestherapy with high doses of steroids and cytotoxic drugs such ascyclophosphamide. (B. L. Kotzin [1996]).

The serological hallmark of SLE, and the primary diagnostic testavailable, is elevated serum levels of IgG antibodies to constituents ofthe cell nucleus, such as double-stranded DNA (dsDNA), single-strandedDNA (ss-DNA), and chromatin. Among these autoantibodies, IgG anti-dsDNAantibodies play a major role in the development of lupusglomerulonephritis (GN). (B. H. Hahn and B. Tsao, Antibodies to DNA, In:Dubois' Lupus Erythematosus, 4th ed., D. J. Wallace and B. Hahn, eds.,Lea and Febiger, Philadelphia, pp. 195-201 [1993]; Ohnishi et al.,Comparison of pathogenic and nonpathogenic murine antibodies to DNA:Antigen binding and structural characteristics, Int. Immunol. 6:817-30[1994]). Glomerulonephritis is a serious condition in which thecapillary walls of the kidney's blood purifying glomeruli becomethickened by accretions on the epithelial side of glomerular basementmembranes. The disease is often chronic and progressive and may lead toeventual renal failure.

The mechanisms by which autoantibodies are induced in these autoimmunediseases remains unclear. As there has been no known cause of SLE, towhich diagnosis and/or treatment could be directed, treatment has beendirected to suppressing immune responses, for example with macrolideantibiotics, rather than to an underlying cause. (E.g., Hitoshi et al.,Immunosuppressive agent, U.S. Pat. No. 4,843,092).

Another disorder for which immunosuppression has been tried is Crohn'sdisease. Crohn's disease symptoms include intestinal inflammation andthe development of intestinal stenosis and fistulas; neuropathy oftenaccompanies these symptoms. Anti-inflammatory drugs, such as5-aminosalicylates (e.g., mesalamine) or corticosteroids, are typicallyprescribed, but are not always effective. (Reviewed in V. A. Botoman etal., Management of Inflammatory Bowel Disease, Am. Fam. Physician57(1):57-68 [1998]). Immunosuppression with cyclosporine is sometimesbeneficial for patients resistant to or intolerant of corticosteroids.(J. Brynskov et al., A placebo-controlled, double-blind, randomizedtrial of cyclosprorine therapy in active chronic Crohn's disease, N.Engl. J. Med. 321(13):845-50 [1989]).

Nevertheless, surgical correction is eventually required in 90% ofpatients; 50% undergo colonic resection. (K. Leiper et al.,Adjuvantpost-operative therapy, Baillieres Clin. Gastroenterol. 12(1):179-99[1998]; F. Makowiec et al., Long-term follow-up after resectionalsurgery in patients with Crohn's disease involving the colon, Z.Gastroenterol. 36(8):619-24 [1998]). The recurrence rate after surgeryis high, with 50% requiring further surgery within 5 years. (K. Leiperet al. [1998]; M. Besnard et al., Postoperative outcome of Crohn'sdisease in 30 children, Gut 43(5):634-38 [1998]).

One hypothesis for the etiology of Crohn's disease is that a failure ofthe intestinal mucosal barrier, possibly resulting from geneticsusceptibilities and environmental factors (e.g., smoking), exposes theimmune system to antigens from the intestinal lumen including bacterialand food antigens (e.g., Söderholm et al, Epithelial permeability toproteins in the non-inflamed ileum of Crohn's disease?, Gastroenterol.117:65-72 [1999]; D. Hollander et al., Increased intestinal permeabilityin patients with Crohn's disease and their relatives. A possibleetiologic factor, Ann. Intern. Med. 105:883-85 [1986]; D. Hollander, Theintestinal permeability barrier. A hypothesis to its involvement inCrohn's disease, Scand. J. Gastroenterol. 27:721-26 [1992]). Anotherhypothesis is that persistent intestinal infection by pathogens such asMycobacterium paratuberculosis, Listeria monocytogenes, abnormalEscherichia coli, or paramyxovirus, stimulates the immune response; oralternatively, symptoms result from a dysregulated immune response toubiquitous antigens, such as normal intestinal microflora and themetabolites and toxins they produce. (R. B. Sartor, Pathogenesis andImmune Mechanisms of Chronic Inflammatory Bowel Diseases, Am. J.Gastroenterol. 92(12):5S-11S [1997]). The presence of IgA and IgGanti-Sacccharomyces cerevisiae antibodies (ASCA) in the serum was foundto be highly diagnostic of pediatric Crohn's disease. (F. M. Ruemmele etal., Diagnostic accuracy of serological assays in pediatric inflammatorybowel disease, Gastroenterol. 115(4):822-29 [1998]; E. J. Hoffenberg etal., Serologic testing for inflammatory bowel disease, J. Pediatr.134(4):447-52 [1999]).

In Crohn's disease, a dysregulated immune response is skewed towardcell-mediated immunopathology. (S. I. Murch, Local and systemic effectsof macrophage cytokines in intestinal inflammation, Nutrition 14:780-83[1998]). But immunosuppressive drugs, such as cyclosporine, tacrolimus,and mesalamine have been used to treat corticosteroid-resistant cases ofCrohn's disease with mixed success. (J. Brynskov et al. [1989]; K.Fellerman et al., Steroid-unresponsive acute attacks of inflammatorybowel disease: immunomodulation by tacrolimus[FK506], Am. J.Gastroenterol. 93(10):1860-66 [1998]).

Recent efforts to develop diagnostic and treatment tools against Crohn'sdisease have focused on the central role of cytokines. (S. Schreiber,Experimental immunomodulatory therapy of inflammatory bowel disease,Neth. J. Med. 53(6):S24-31 [1998]; R. A. van Hogezand and H. W.Verspaget, The future role of anti-tumour necrosis factor-alpha productsin the treatment of Crohn's disease, Drugs 56(3):299-305 [1998]).Cytokines are small secreted proteins or factors (5 to 20 kD) that havespecific effects on cell-to-cell interactions, intercellularcommunication, or the behavior of other cells. Cytokines are produced bylymphocytes, especially T_(H)1 and T_(H)2 lymphocytes, monocytes,intestinal macrophages, granulocytes, epithelial cells, and fibroblasts.(Reviewed in G. Rogler and T. Andus, Cytokines in inflammatory boweldisease, World J. Surg. 22(4):382-89 [1998]; H. F. Galley and N. R.Webster, The immuno-inflammatory cascade, Br. J. Anaesth. 77:11-16[1996]). Some cytokines are pro-inflammatory (e.g., tumor necrosisfactor [TNF]-α, interleukin [IL]-1(α and β), IL-6, IL-8, IL-12, orleukemia inhibitory factor [LIF]); others are anti-inflammatory (e.g.,IL-1 receptor antagonist [IL-1ra], IL-4, IL-10, IL-11, and transforminggrowth factor [TGF]-β). However, there may be overlap and functionalredundancy in their effects under certain inflammatory conditions.

In active cases of Crohn's disease, elevated concentrations of TNF-α andIL-6 are secreted into the blood circulation, and TNF-α, IL-1, IL-6, andIL-8 are produced in excess locally by mucosal cells. (Id.; K. Funakoshiet al., Spectrum of cytokine gene expression in intestinal mucosallesions of Crohn's disease and ulcerative colitis, Digestion 59(1):73-78[1998]). These cytokines can have far-ranging effects on physiologicalsystems including bone development, hematopoiesis, and liver, thyroid,and neuropsychiatric function. Also, an imbalance of the IL-1β/IL-1raratio, in favor of pro-inflammatory IL-1β, has been observed in patientswith Crohn's disease. (G. Rogler and T. Andus [1998]; T. Saiki et al.,Detection of pro- and anti-inflammatory cytokines in stools of patientswith inflammatory bowel disease, Scand. J. Gastroenterol. 33(6):616-22[1998]; S. Dionne et al., Colonic explant production of IL-1 and itsreceptor antagonist is imbalanced in inflammatory bowel disease (IBD),Clin. Exp. Imunol. 112(3):435-42 [1998]; But see S. Kuboyama. Increasedcirculating levels of Interleukin-1 receptor antagonist in patients withinflammatory bowel disease, Kurume Med. J. 45(1):33-37 [1998]). Onestudy suggested that cytokine profiles in stool samples could be auseful diagnostic tool for Crohn's disease. (T. Saiki et al [1998]).

Treatments that have been proposed for Crohn's disease include the useof various cytokine antagonists (e.g., IL-1ra), inhibitors (e.g., ofIL-1β converting enzyme and antioxidants) and anti-cytokine antibodies.(G. Rogler and T. Andus [1998]; R. A. van Hogezand and H. W. Verspaget[1998]; J. M. Reimund et al., Antioxidants inhibit the in vitroproduction of inflammatory cytokines in Crohn's disease and ulcerativecolitis, Eur. J. Clin. Invest. 28(2):145-50 [1998]; N. Lugering et al.,Current concept of the role of monocytes/macrophages in inflammatorybowel disease-balance of pro-inflammatory and immunosuppressivemediators, Ital. J. Gastroenterol. Hepatol. 30(3):338-44 [1998]; M. E.McAlindon et al., Expression of interleukin 1 beta and interleukin 1beta converting enzyme by intestinal macrophages in health andinflammatory bowel disease, Gut 42(2):214-19 [1998]). In particular,monoclonal antibodies against TNF-α have been tried with some success inthe treatment of Crohn's disease. (S. R. Targan et al., A short-termstudy of chimeric monoclonal antibody cA2 to tumor necrosis factor alphafor Crohn's disease. Crohn's Disease cA2 Study Group, N. Engl. J. Med.337(15):1029-35 [1997]; W. A. Stack et al., Randomised controlled trialof CDP571 antibody to tumour necrosis factor-alpha in Crohn's disease,Lancet 349(9051):521-24 [1997]; H. M. van Dullemen et al., Treatment ofCrohn's disease with anti-tumor necrosis factor chimeric monoclonalantibody (cA2), Gastroenterol. 109(1):129-35 [1995]).

Another approach to the treatment of Crohn's disease has focused on atleast partially eradicating the bacterial community that may betriggering the inflammatory response and replacing it with anon-pathogenic community. For example, McCann et al. (McCann et al,Method for treatment of idiopathic inflammatory bowel disease, U.S. Pat.No. 5,599,795) disclosed a method for the prevention and treatment ofCrohn's disease in human patients. Their method was directed tosterilizing the intestinal tract with at least one antibiotic and atleast one anti-fungal agent to kill off the existing flora and replacingthem with different, select, well-characterized bacteria taken fromnormal humans. Borody taught a method of treating Crohn's disease by atleast partial removal of the existing intestinal microflora by lavageand replacement with a new bacterial community introduced by fecalinoculum from a disease-screened human donor or by a compositioncomprising Bacteroides and Escherichia coli species. (T. J. Barody,Treatment of gastro-intestinal disorders with a fecal composition or acomposition of bacteroides and E. coli, U.S. Pat. No. 5,443,826).However, there has been no known cause of Crohn's disease to whichdiagnosis and/or treatment could be directed.

Pain is a common symptom associated with irritable bowel syndrome,fibromyalgia, chronic fatigue syndrome, depression, ADHD, autoimmunediseases, and Crohn's disease. While the experience of pain isintertwined with a person's emotions, memory, culture, and psychosocialsituation (D. A. Drossman and W. G. Thompson, Irritable bowel syndrome:a graduated, multicomponent treatment approach, Ann. Intern. Med.116:1009-16 [1992]), evidence shows that certain cytokinemediated-immune responses can influence the perception of pain.Cytokines can be released in response to a variety of irritants and canmodulate the perception of pain. For example, exposure of humanbronchial epithelial cells to irritants, including acidic pH, results ina receptor-mediated release of inflammatory cytokines IL-6, IL-8, andTNF-α. (B. Veronesi et al., Particulate Matter initiates inflammatorycytokine release by activation of capsaicin and acid receptors in ahuman bronchial epithelial cell line, Toxicol. Appl. Pharmacol.154:106-15 [1999]). Irritant receptors on cell surfaces, e.g., receptorssensitive to noxious stimuli, such as capsaicin and pH, mediate therelease of cytokines and also mediate the release of neuropeptides fromsensory nerve fibers, which is known to result in a neurogenicinflammatory processes and hyperalgesia (excessive sensitivity to pain).(Id.; R.O.P. de Campos et al., Systemic treatment with Mycobacteriumbovis bacillus calmett-guerin (BCG) potentiates kinin B ₁ receptoragonist-induced nociception and oedema formation in the formalin test inmice, Neuropeptides 32(5):393-403 [1998]).

The perception of pain, is also influenced by the mediation of kinin B₁and B₂ receptors which bind peptides called kinins, e.g., thenonapeptide bradykinin or the decapeptide kallidin (lysyl bradykinin).While the precise mechanism of action is unknown, kinins cause therelease of other pro-inflammatory and hyperalgesic mediators such asneuropeptides. Cytokines IL-1 (α and β), IL-2, IL-6, and TNF-α arethought to activate kinin B₁ receptor, and thus can contribute toenhanced perception of pain. (R.O.P. de Campos et al. [1998]. Theendotoxin of Escherichia coli significantly activated kinin B₁receptor-mediated neurogenic and inflammatory pain responses in animals.(M. M. Campos et al., Expression of B ₁ kinin receptors mediating pawoedema formalin-induced nociception. Modulation by glucocorticoids, Can.J. Physiol. Pharmacol. 73:812-19 [1995]).

It has also been shown that IL-1β, IL-6, and TNF-α, administered to themammalian brain, can modulate pain perception viaprostaglandin-dependent processes. (T. Hori et al., Pain modulatoryactions of cytokines and prostaglandin E ₂ in the Brain, Ann. N.Y. Acad.Sci. 840:269-81 [1998]). Granulocytes, which accumulate in nearly allforms of inflammation, are non-specific amplifiers and effectors ofspecific immune responses, and they can also modulate the perception ofpain. Neutrophils, a type of granulocyte cell, are known to accumulatein response to IL-1β, and neutrophil accumulation plays a crucialpositive role in the development of nerve growth factor (NGF)-inducedhyperalgesia. (G. Bennett et al, Nerve growth factor inducedhyperalgesia in the rat hind paw is dependent on circulatingneutrophils, Pain 77(3):315-22 [1998]; see also E. Feher et al., Directmorphological evidence of neuroimmunomodulation in colonic mucosa ofpatients with Crohn's disease, Neuroimmunomodulation 4(5-6):250-57[1997]).

Hyperalgesia (visceral, musculoskeletal, and/or cutaneous) is a commonclinical observation in IBS and fibromyalgia. As many as 60% of subjectswith IBS have reduced sensory thresholds for rectal distension. (H.Mertz et al., Altered rectal perception is a biological marker ofpatients with the irritable bowel syndrome, Gastroenterol.109:40-52[1995]). While the etiology for this hyperalgesia has remained elusive,it has been hypothesized that there is a sensitization of afferentpathways in IBS. (E. A. Mayer et al., Basic and clinical aspects ofvisceral hyperalgesia, Gastroenterol 1994;107:271-93 [1994]; L. Bueno etal., Mediators and pharmacology of visceral sensitivity: from basic toclinical investigations, Gastroenterol. 112:1714-43 [1997]).Fibromyalgia is, by definition; a hyperalgesic state since the AmericanCollege of Rheumatology defines fibromyalgia as a history of global painin the setting of 11 out of 18 predefined tender points. (F. Wolfe etal., The American College of Rheumatology 1990 criteria for theclassification of fibromyalgia, Arthritis Rheum. 33:160-72 [1990]).Evidence implies that the hyperalgesia of fibromyalgia is not simplytrigger point-related but rather a global hyperalgesia. (L. Vecchiet etal., Comparative sensory evaluation of parietal tissues in painful andnonpainful areas in fibromyalgia and myofascial pain syndrome, In:Gebhart G F, Hammond D L, Jensen T S, editors, Progress in Pain Researchand Management, Vol.2, Seattle: IASP Press, pp. 177-85 [1994]; J.Sorensen et al., Hyperexcitability in fibromyalgia, J. Rheumatol.25:152-55 [1998]).

While hyperalgesia has not been clearly shown to be associated withCrohn's disease (C. N. Bernstein et al., Rectal afferent function inpatients with inflammatory and functional intestinal disorders, Pain66:151-61 [1996]), cytokine and neuropeptide levels are altered in IBS,fibromyalgia, and Crohn's disease. Indirect evidence forhypersensitivity in Crohn's disease is suggested by elevated TNF-α andsubstance P receptor levels (C. R. Mantyh et al., Receptor binding sitesfor substance P, but not substance K or neuromedin K, are expressed inhigh concentrations by arterioles, venules, and lymph nodules insurgical specimens obtained from patients with ulcerative colitis andCrohn's disease, Proc. Natl. Acad. Sci. 85:3235-39 [1988]; S. Mazumdarand K. M. Das, Immunocytochemical localization of vasoactive intestinalpeptide and substance P in the colon from normal subjects and patientswith inflammatory bowel disease, Am. J. Gastrol. 87:176-81 [1992]; C. R.Mantyh et al., Differential expression of substance P receptors inpatients with Crohn's disease and ulcerative colitis, Gastroenterol.1995;109:850-60 [1995]), which have been shown to be associated withhypersensitivity. It has been shown that levels of substance P, aneuropeptide, are elevated in the cerebrospinal fluid of subjects withfibromyalgia (H. Vaeroy et al., Elevated CSF levels of substance P andhigh incidence of Raynaud's phenomenon in patients with fibromyalgia:new features for diagnosis, Pain 32:21-26 [1988]; I. J. Russell et al.,Elevated cerebrospinal fluid levels of substance P in patients with thefibromyalgia syndrome, Arthritis Rheum. 37:1593-1601 [1994]), and anincrease in substance P-sensitive nerve endings has been observed insubjects with IBS. (X. Pang et al., Mast cell substance P-positive nerveinvolvement in a patient with both irritable bowel syndrome andinterstitial cystitis, Urology 47:436-38 [1996]).

Mental functioning and feelings of fatigue or depression can also beinfluenced by immune responses. Peripherally released pro-inflammatorycytokines, such as IL-1, IL-6 and TNF-α, act on brain cellular targetsand have been shown to depress spontaneous and learned behavior inanimals; the vagus nerve has been shown to mediate the transmissions ofthe immune message to the brain, resulting in production ofpro-inflammatory cytokines centrally in the brain. (R. Dantzer et al.,Cytokines and sickness behavior, Ann. N.Y. Acad. Sci. 840:586-90[1998]). In addition, there is bidirectional interplay betweenneurotransmitters and the immune system; lymphocytes and macrophagesbear surface receptors for the stress hormone corticotrophin releasinghormone (CRH), and they respond to CRH by enhanced lymphocyteproliferation and feedback upregulation of hypothalamic CRH production.(S. H. Murch [1998]).

Pituitary production of proopiomelanocortins, such as endorphins andenkephalins, is upregulated by IL-1 and IL-2, possibly mediated by CRH,and lymphocytes and macrophages recognize these endogenous opiates viasurface receptors. (S. H. Murch [1998]). Lymphocytes (T_(H)2) andmacrophages also produce and process enkephalin to an active form.Macrophage-derived cytokines, such as TNF-α, IL-1, and IL-6, are knownto modulate neurotransmitter release and to affect overall neuralactivity; cytokines can induce classic illness behavior such assomnolence, apathy, depression, irritability, confusion, poor memory,impaired mental concentration, fever and anorexia.

While immunological responses can lead to symptoms of irritable bowelsyndrome, fibromyalgia, chronic fatigue syndrome, depression, ADHD,autoimmune diseases, and Crohn's disease, there has been a definite needto determine a causal factor, for each of these diagnostic categories,to which diagnostic testing and treatment can be directed.

No association has ever been made between any of the afore-goingdiagnostic categories and small intestinal bacterial overgrowth (SIBO).SIBO, also known as small bowel bacterial overgrowth (SBBO), is anabnormal condition in which aerobic and anaerobic enteric bacteria fromthe colon proliferate in the small intestine, which is normallyrelatively free of bacterial contamination. SIBO is defined as greaterthan 10⁶ CFU/mL small intestinal effluent (R. M. Donaldson, Jr., Normalbacterial populations of the intestine and their relation to intestinalfunction, N. Engl. J. Med. 270:938-45 [1964]). Typically, the symptomsinclude abdominal pain, bloating, gas and alteration in bowel habits,such as constipation and diarrhea.

SIBO has, until recently, mostly been suspected in subjects withsignificant malabsorptive sequelae. Most of the described cases of SIBOinvolve anatomic alterations such as physical obstruction (E. A. Deitchet al., Obstructed intestine as a reservoir for systemic infection, Am.J. Surg. 159:394 [1990]), surgical changes (e.g., L. K. Enander et al.The aerobic and anaerobic microflora of the gastric remnant more than 15years after Billroth II resection, Scand. J. Gastroenterol. 17:715-20[1982]), direct communication of the small intestine with coloniccontents such as fistulae (O. Bergesen et al., Is vitamin B12malabsorption in bile fistula rats due to bacterial overgrowth? A studyof bacterial metabolic activity in the small bowel, Scand. J.Gastroenterol. 23:471-6 [1988]) and ileocecal valve dysfunction(surgical or otherwise) (W. O. Griffin, Jr, et al., Prevention of smallbowel contamination by ileocecal valve, S. Med. J.64: 1056-8 [1971]; P.Rutgeerts et al., Ileal dysfunction and bacterial overgrowth in patientswith Crohn's disease, Eur. J. Clin. Invest. 11:199-206 [1981]). Lesscommonly, SIBO has been associated with chronic pancreatitis (E. Trespiand A. Ferrieri, Intestinal bacterial overgrowth during chronicpancreatitis, Curr. Med. Res. Opin. 15:47-52 [1999]), hypochlorhydria(e.g., S. P. Pereira et al, Drug-induced hypochlorhydria causes highduodenal bacterial counts in the elderly, Aliment. Pharmacol.Ther.12:99-104 [1998]), and immunodeficiency (C. Pignata et al., Jejunalbacterial overgrowth and intestinal permeability in children withimmunodeficiency syndromes, Gut 31:879-82 [1990]; G. M. Smith et al.,Small intestinal bacterial overgrowth in patients with chroniclymphocytic leukemia, J. Clin. Pathol. 43:57-9 [1990]).

SIBO has been associated with infections of the abdominal cavity incases of alcoholic cirrhosis. (F. Casafont Morencos et al., Small bowelbacterial overgrowth in patients with alcoholic cirrhosis, Dig. Dis.Sci. 40(6):1252-1256 [1995]; J. Chesta et al., Abnormalities in proximalsmall bowel motility in patients with cirrhosis, Hepatology 17(5):828-32[1993]; C. S. Chang et al., Small intestine dysmotility and bacterialovergrowth in cirrhotic patients with spontaneous bacterial peritonitis,Hepatology 28(5):1187-90 [1998]). SIBO has also been associated withsymptoms of chronic diarrhea, anorexia or nausea in elderly patients,and the prevalence of overgrowth in subjects over 75 years old isreported to be as high as 79% even in the absence of clinically evidentclues of overgrowth or achlorhydria (S. M. Riordan et al., Smallintestinal bacterial overgrowth in the symptomatic elderly, Am. J.Gastroenterol. 92(1):47-51 [1997]). SIBO is also associated with chronicdigestive symptoms in children, especially infants under two years ofage (D. De Boissieu et al., Small-bowel bacterial overgrowth in childrenwith chronic digestive diarrhea, abdominal pain, or both, J. Pediatr.128(2):203-07 [1996]), and with chronic diarrhea after livertransplantation in children. (D. R. Mack et al., Small bowel bacterialovergrowth as a cause of chronic diarrhea after liver transplantation inchildren, Liver Transpl. Surg. 4(2):166-69 [1998]).

Although diabetic enteropathy (F. Goldstein et al., Diabetic diarrheaand steatorrhea. Microbiologic and clinical observations, Ann. Intern.Med. 1970;72:215-8 [1970]), idiopathic intestinal pseudo-obstruction (A.J. Pearson et al., Intestinal pseudo-obstruction with bacterialovergrowth in the small intestine, Am . J. Dig. Dis. 14:200-05 [1969])and scleroderma (I. J. Kahn et al., Malabsorption in intestinalscleroderma: Correction with antibiotics, N. Engl. J. Med. 274: 1339-44[1966]) are all known to produce motility disturbances leading to SIBO.Two previous reports have examined small bowel motility amonganatomically and medically naive SIBO subjects. (G. Vantrappen et al.,The interdigestive motor complex of normal subjects and patients withbacterial overgrowth of the small intestine, J. Clin. Invest. 59:1158-66 [1977]; P. O. Stotzer et al., Interdigestive and postprandialmotility in small-intestinal bacterial overgrowth, Scand. J.Gastroenterol. 31:875-80 [1996]). These authors suggest that themajority of subjects with SIBO in the absence of other predisposingconditions, lack the phase III of interdigestive motility during shortterm recordings.

Phase III of interdigestive motility is a period of phasic contractionspropagating through the length of the small intestine, approximatelyonce every 87.2±5.4 minutes in the fasting state. (E. E. Soffer et al.,Prolonged ambulatory duodeno-jejunal manometry in humans: Normal valuesand gender effect, Am. J. Gastrol. 93:1318-23 [1998]). This fastingevent is responsible for sweeping residue including small bowelcontaminants, such as accumulated bacteria, into the colon inpreparation for the next meal. (V. B. Nieuwenhujuijs et al., The role ofinterdigestive small bowel motility in the regulation of gut microflora,bacterial overgrowth, and bacterial translocation in rats, Ann. Surg.228: 188-93 [1998]; E. Husebye, Gastrointestinal motility disorders andbacterial overgrowth, J. Intem. Med. 237:419-27 [1995]). The endogenouspeptide, motilin, is involved in the mediation of this event. (G.Vantrappen et al., Motilin and the interdigestive migrating motorcomplex in man, Dig. Dis. Sci. 24:497-500 (1979]). Other prokineticagents, such as erythromycin, are believed to act on the motilinreceptor and have been shown to rapidly induce an interdigestivemotility event in dogs and humans. (M. F. Otterson and S. K. Sarna,Gastrointestinal motor effect of erythromycin, Am. J. Physiol.259:G355-63; T. Tomomasa et al., Erythromycin induces migrating motorcomplex in human gastrointestinal tract, Dig. Dis. Sci. 31:157-61[1986]).

There remains a need for an underlying causal factor, to whichdiagnostic testing and treatment can be directed, for irritable bowelsyndrome; fibromyalgia; chronic fatigue syndrome; depression; ADHD; MS,SLE and other autoimmune diseases; and Crohn's disease. This and otherbenefits of the present invention are described herein.

SUMMARY OF THE INVENTION

The present invention relates to the diagnosis or treatment of irritablebowel syndrome (IBS); fibromyalgia (FM); chronic fatigue syndrome (CFS);depression; attention deficit/hyperactivity disorder (ADHD); multiplesclerosis (MS), systemic lupus erythematosus (SLE) and other autoimmunediseases; and Crohn's disease (CD). Specifically, the present methodsare based on the detection and treatment of a unified cause for all ofthem, i.e., small intestinal bacterial overgrowth (SIBO).

The method of diagnosing irritable bowel syndrome, fibromyalgia, chronicfatigue syndrome, depression, ADHD, autoimmune diseases, or Crohn'sdisease involves detecting the presence of small intestinal bacterialovergrowth in a human subject having at least one symptom associatedwith a suspected diagnosis of any of those diagnostic categories.

The present invention also relates to a method of treatment forirritable bowel syndrome, fibromyalgia, chronic fatigue syndrome,depression, ADHD, autoimmune diseases, or Crohn's disease involving atherapeutic regime to at least partially eradicate small intestinalbacterial overgrowth. This therapy regime can include treatment withanti-microbial agents or other therapeutic approaches to at leastpartially eradicating bacterial overgrowth, e.g., lavage, probiotictechniques, or by normalizing or increasing intestinal phase IIIinterdigestive motility with, for example, administration of a modifieddiet or a chemical prokinetic agent. The method improves symptoms,including hyperalgesia related to SIBO and disorders caused by SIBO,such as irritable bowel syndrome, fibromyalgia, and Crohn's disease.

The present invention also relates to kits for the diagnosis andtreatment of irritable bowel syndrome, fibromyalgia, chronic fatiguesyndrome, depression. ADHD, autoimmune diseases, or Crohn's disease.

These and other advantages and features of the present invention will bedescribed more fully in a detailed description of the preferredembodiments which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows visual analog scores reported by subjects with IBS and SIBObefore and after antibiotic treatment.

FIG. 2 shows visual analog scores from subjects with IBS and SIBO in apilot study, before and after antibiotic treatment.

FIG. 3 shows visual analog scores reported by subjects with fibromyalgiaand SIBO before and after antibiotic treatment.

FIG. 4 shows the correlation between the degree of improvement insymptoms and residual breath hydrogen production after antibiotictreatment in subjects with fibromyalgia and SIBO.

FIG. 5 shows visual analog scores reported by subjects with Crohn'sdisease and SIBO before and after antibiotic treatment.

FIG. 6 shows the correlation between degree of improvement in symptomsand residual breath hydrogen production after antibiotic treatment insubjects with Crohn's disease.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to method of diagnosing irritable bowelsyndrome, fibromyalgia, chronic fatigue syndrome, depression, ADHD, anautoimmune disease, such as multiple sclerosis or systemic lupuserythematosus, or Crohn's disease. The method involves detecting thepresence of small intestinal bacterial overgrowth in a human subject whohas at least one symptom associated with a suspected diagnosis of anyone of these diagnostic categories.

In accordance with the method, the detection of SIBO in the humansubject corroborates the suspected diagnosis held by a qualified medicalpractitioner who, prior to the detection of SIBO in the human subject,suspects from more limited clinical evidence that the human subject hasirritable bowel syndrome, fibromyalgia, chronic fatigue syndrome.depression, ADHD, an autoimmune disease, or Crohn's disease. By applyingthe present diagnostic method the suspected diagnosis is corroborated,i.e., confirmed, sustained, substantiated, supported, evidenced,strengthened, affirmed or made more firm.

The skilled medical practitioner is aware of suitable up-to-datediagnostic criteria by which a suspected diagnosis is reached. Thesediagnostic criteria are based on a presentation of symptom(s) by a humansubject. For example, these criteria include, but are not limited to,the Rome criteria for IBS (W. G. Thompson, Irritable bowel syndrome:pathogenesis and management, Lancet 341:1569-72 [1993]) and the criteriafor CFS established by the Centers for Disease Control and Prevention(CDC). (K. Fukuda et al., The chronic fatigue syndrome: a comprehensiveapproach to its definition and study, Ann. Intem. Med. 121:953-59[1994]). The diagnostic criteria for fibromyalgia of the AmericanCollege of Rheumatology will also be familiar (F. Wolfe et al., TheAmerican College of Rheumatology 1990 Criteria for the Classification ofFibromyalgia: Report of the Multicenter Criteria Committee, ArthritisRheum. 33:160-72 [1990]), as will be the criteria for depression or ADHDprovided for example, by the Diagnostic and Statistical Manual (DSM)-IVor its current version. (E.g., G. Tripp et al., DSM-IV and ICD-10: acomparison of the correlates of ADHD and hyperkinetic disorder, J. Am.Acad. Child Adolesc. Psychiatry 38(2):156-64 [1999]). Symptoms ofsystemic lupus erythematosus include the 11 revised criteria of theAmerican College of Rheumatology, such as a typical malar or discoidrash, photosensitivity, oral ulcers, arthritis, serositis, or disordersof blood, kidney or nervous system. (E. M Tan et al., The 1982 revisedcriteria for the classification of systemic lupus erythematosus [SLE],Arthritis Rheum. 25:1271-77 [1982]). Appropriate diagnostic criteria formultiple sclerosis are also familiar (e.g., L. A. Rolak, The diagnosisof multiple sclerosis, Neuronal Clin. 14(1):27-43 [1996]), as aresymptoms of Crohn's disease useful in reaching a suspected diagnosis.(e.g., J. M. Bozdech and R. G. Farmer, Diagnosis of Crohn's disease,Hepatogastroenterol. 37(1):8-17 [1990]; M. Tanaka and R. H. Riddell, Thepathological diagnosis and differential diagnosis of Crohn's disease,Hepatogastroenterol. 37(1):18-31 [1990]; A. B. Price and B. C. Morson,Inflammatory bowel disease: the surgical pathology of Crohn's diseaseand ulcerative colitis, Hum. Pathol. 6(1):7-29 [1975]). The practitioneris, of course not limited to these illustrative examples for diagnosticcriteria, but should use criteria that are current.

Detecting the presence of small intestinal bacterial overgrowth (i.e.,SIBO) is accomplished by any suitable method. For example, one preferredmethod of detecting SIBO is breath hydrogen testing. (E.g., P. Kerlinand L. Wong, Breath hydrogen testing in bacterial overgrowth of thesmall intestine, Gastroenterol. 95(4):982-88 [1988]; A. Strocchi et al.,Detection of malabsorption of low doses of carbohydrate: accuracy ofvarious breath H ₂ criteria, Gastroenterol. 105(5):1404-1410 [1993]; D.de Boissieu et al., [1996]; P. J. Lewindon et al., Bowel dysfunction incystic fibrosis: importance of breath testing, J. Paedatr. Child Health34(1):79-82 [1998]). Breath hydrogen or breath methane tests are basedon the fact that many obligately or facultatively fermentative bacteriafound in the gastrointestinal tract produce detectable quantities ofhydrogen or methane gas as fermentation products from a substrateconsumed by the host, under certain circumstances. Substrates includesugars such as lactulose, xylose, lactose, or glucose. The hydrogen ormethane produced in the small intestine then enters the blood stream ofthe host and are gradually exhaled.

Typically, after an overnight fast, the patient swallows a controlledquantity of a sugar, such as lactulose, xylose, lactose, or glucose, andbreath samples are taken at frequent time intervals, typically every 10to 15 minutes for a two- to four-hour period. Samples are analyzed bygas chromatography or by other suitable techniques, singly or incombination. Plots of breath hydrogen in patients with SIBO typicallyshow a double peak, i.e., a smaller early hydrogen peak followed by alarger hydrogen peak, but a single hydrogen peak is also a usefulindicator of SIBO, if peak breath hydrogen exceeds the normal range ofhydrogen for a particular testing protocol. (See, G. Mastropaolo and W.D. Rees, Evaluation of the hydrogen breath test in man: definition andelimination of the early hydrogen peak, Gut 28(6):721-25 [1987]).

A variable fraction of the population fails to exhale appreciablehydrogen gas during intestinal fermentation of lactulose; the intestinalmicroflora of these individuals instead produce more methane. (G.Corazza et al., Prevalence and consistency of low breath H ₂ excretionfollowing lactulose ingestion. Possible implications for the clinicaluse of the H ₂ breath test, Dig. Dis. Sci. 38(11):2010-16 [1993]; S. M.Riordan et al., The lactulose breath hydrogen test and small intestinalbacterial overgrowth, Am. J. Gastroentrol. 91(9); 1795-1803 [1996]).Consequently, in the event of an initial negative result for breathhydrogen, or as a precaution, methane and/or carbon dioxide contents ineach breath sample are optionally measured, as well as hydrogen, or asubstrate other than lactulose is optionally used. Also, acting as acheck, the presence of SIBO is demonstrated by a relative decrease inpeak hydrogen exhalation values for an individual subject afterantimicrobial treatment, in accordance with the present invention,compared to pretreatment values.

Another preferred method of detecting bacterial overgrowth is by gaschromatography with mass spectrometry and/or radiation detection tomeasure breath emissions of isotope-labeled carbon dioxide, methane, orhydrogen, after administering an isotope-labeled substrate that ismetabolizable by gastrointestinal bacteria but poorly digestible by thehuman host, such as lactulose, xylose, mannitol, or urea. (E.g., G. R.Swart and J. W. van den Berg, ¹³ C breath test in gastrointestinalpractice, Scand. J. Gastroenterol. [Suppl.] 225:13-18 [1998]; S. F.Dellert et al., The 13C-xylose breath test for the diagnosis of smallbowel bacterial overgrowth in children, J. Pediatr. Gastroenterol. Nutr.25(2):153-58 [1997]; C. E. King and P. P. Toskes, Breath tests in thediagnosis of small intestinal bacterial overgrowth, Crit. Rev. Lab. Sci.21(3):269-81 [1984]). A poorly digestible substrate is one for whichthere is a relative or absolute lack of capacity in a human forabsorption thereof or for enzymatic degradation or catabolism thereof.

Suitable isotopic labels include ¹³C or ¹⁴C. For measuring methane orcarbon dioxide, suitable isotopic labels can also include ²H and ³H or¹⁷O and ¹⁸O, as long as the substrate is synthesized with the isotopiclabel placed in a metabolically suitable location in the structure ofthe substrate, i.e., a location where enzymatic biodegradation byintestinal microflora results in the isotopic label being sequestered inthe gaseous product. If the isotopic label selected is a radioisotope,such as ¹⁴C, ³H, or ¹⁵O, breath samples can be analyzed by gaschromatography with suitable radiation detection means. (E.g., C. S.Chang et al., Increased accuracy of the carbon-14 D-xylose breath testin detecting small-intestinal bacterial overgrowth by correction withthe gastric emptying rate, Eur. J. Nucl. Med. 22(10):1118-22 [1995]; C.E. King and P. P. Toskes, Comparison of the 1-gram [ ¹⁴ C]xylose,10-gram lactulose-H ₂ , and 80-gram glucose-H ₂ breath tests in patientswith small intestine bacterial overgrowth, Gastroenterol. 91(6):1447-51[1986]; A. Schneider et al., Value of the ¹⁴ C-D-xylose breath test inpatients with intestinal bacterial overgrowth, Digestion 32(2):86-91[1985]).

Another preferred method of detecting small intestinal bacterialovergrowth is direct intestinal sampling from the human subject. Directsampling is done by intubation followed by scrape, biopsy, or aspirationof the contents of the intestinal lumen, including the lumen of theduodenum, jejunum, or ileum. The sampling is of any of the contents ofthe intestinal lumen including material of a cellular, fluid, fecal, orgaseous nature, or sampling is of the lumenal wall itself. Analysis ofthe sample to detect bacterial overgrowth is by conventionalmicrobiological techniques including microscopy, culturing, and/or cellnumeration techniques.

Another preferred method of detecting small intestinal bacterialovergrowth is by endoscopic visual inspection of the wall of theduodenum, jejunum, and/or ileum.

The preceding are merely illustrative and non-exhaustive examples ofmethods for detecting small intestinal bacterial overgrowth.

The present invention also relates to a method of treating irritablebowel syndrome, fibromyalgia, chronic fatigue syndrome, depression,ADHD, an autoimmune disease, or Crohn's disease. The treatment methodinvolves detecting the presence of small intestinal bacterial overgrowthin a human subject, in accordance with the diagnostic method describedabove, and at least partially eradicating the bacterial overgrowth.After the SIBO condition is at least partially eradicated, typicallywithin a couple of weeks, there is an improvement in the symptom(s) ofirritable bowel syndrome, fibromyalgia, chronic fatigue syndrome,depression, ADHD, an autoimmune disease, or Crohn's disease. It is abenefit of the present treatment method that after treatment, subjectsroutinely report feeling better than they have felt in years.

At least partially eradicating the bacterial overgrowth is accomplishedby any suitable method. Most preferably, at least partially eradicatingthe bacterial overgrowth is accomplished by administering anantimicrobial agent, including but not limited to a natural, synthetic,or semi-synthetic antibiotic agent. For example, a course of antibioticssuch as, but not limited to, neomycin, metronidazole, teicoplanin,doxycycline, tetracycline, ciprofloxacin, augmentin, cephalexin (e.g.,Keflex), penicillin, ampicillin, kanamycin, rifamycin, rifaximin, orvancomycin, which may be administered orally, intravenously, orrectally. (R. K. Cleary [1998]; C. P. Kelly and J. T. LaMont,Clostridium difficile infection, Annu. Rev. Med. 49:375-90 [1998]; C. M.Reinke and C. R. Messick, Update on Clostridium difficile-inducedcolitis, Part 2, Am. J. Hosp. Pharm. 51(15):1892-1901 [1994]).

Alternatively, an antimicrobial chemotherapeutic agent, such as a 4- or5-aminosalicylate compound is used to at least partially eradicate theSIBO condition. These can be formulated for ingestive, colonic, ortopical non-systemic delivery systems or for any systemic deliverysystems. Commercially available preparations include4-(p)-aminosalicylic acid (i.e., 4-ASA or para-aminosalicylic acid) or4-(p)-aminosalicylate sodium salt (e.g., Nemasol-Sodium® or Tubasal®).5-Aminosalicylates have antimicrobial, as well as anti-inflammatoryproperties (H. Lin and M. Pimentel, Abstract G3452 at Digestive DiseaseWeek, 100^(th) Annual Meeting of the AGA, Orlando, Fla. [1999]), inuseful preparations including 5-aminosalicylic acid (i.e., 5-ASA,mesalamine, or mesalazine) and conjugated derivatives thereof, availablein various pharmaceutical preparations such as Asacol®, Rowasa®,Claversal®, Pentasa®, Salofalk®, Dipentum® (olsalazine), Azulfidine®(SAZ; sulphasalazine), ipsalazine, salicylazobenzoic acid, balsalazide,or conjugated bile acids, such as ursodeoxycholic acid-5-aminosalicylicacid, and others.

Another preferred method of at least partially eradicating smallintestinal bacterial overgrowth, particularly useful when a subject doesnot respond well to oral or intravenous antibiotics or otherantimicrobial agents alone, is administering an intestinal lavage orenema, for example, small bowel irrigation with a balanced hypertonicelectrolyte solution, such as Go-lytely or fleet phosphosodapreparations. The lavage or enema solution is optionally combined withone or more antibiotic(s) or other antimicrobial agent(s). (E.g., J. A.Vanderhoof et al., Treatment strategies for small bowel bacterialovergrowth in short bowel syndrome, J. Pediatr. Gastroenterol. Nutr.27(2):155-60 [1998])

Another preferred method of at least partially eradicating smallintestinal bacterial overgrowth employs a probiotic agent, for example,an inoculum of a lactic acid bacterium or bifidobacterium. (A. S. Naiduet al., Probiotic spectra of lactic acid bacteria, Crit. Rev. Food Sci.Nutr. 39(1):13-126 [1999]; J. A. Vanderhoof et al. [1998]; G. W.Tannock, Probiotic propertyies of lactic acid bacteria: plenty of scopefor R & D, Trends Biotechnol. 15(7):270-74 [1997]; S. Salminen et al.,Clinical uses of probiotics for stabilizing the gut mucosal barrier:successful strains and future challenges, Antonie Van Leeuwenhoek70(2-4):347-58 [1997]). The inoculum is delivered in a pharmaceuticallyacceptable ingestible formulation, such as in a capsule, or for somesubjects, consuming a food supplemented with the inoculum is effective,for example a milk, yoghurt, cheese, meat or other fermentable foodpreparation. Useful probiotic agents include Bifidobacterium sp. orLactobacillus species or strains, e.g., L. acidophilus, L. rhamnosus, L.plantarum, L. reuteri, L. paracasei subsp. paracasei, or L. caseiShirota, (P. Kontula et al., The effect of lactose derivatives onintestinal lactic acid bacteria, J. Dairy Sci. 82(2):249-56 [1999]; M.Alander et al., The effect of probiotic strains on the microbiota of theSimulator of the Human Intestinal Microbial Ecosystem (SHIME), Int. J.Food Microbiol. 46(1):71-79 [1999]; S. Spanhaak et al., The effect ofconsumption of milk fermented by Lactobacillus casei strain Shirota onthe intestinal microflora and immune parameters in humans, Eur. J. Clin.Nutr. 52(12):899-907 [1998]; W. P. Charteris et al., Antibioticsusceptibility of potentially probiotic Lactobacillus species, J. FoodProt. 61(12): 1636-43 [1998]; B. W. Wolf et al., Safety and tolerance ofLactobacillus reuteri supplementation to a population infected with thehuman immunodeficiency virus, Food Chem. Toxicol. 36(12):1085-94 [1998];G. Gardiner et al., Development of a probiotic cheddar cheese containinghuman-derived Lactobacillus paracasei strains, Appl. Environ. Microbiol.64(6):2192-99 [1998]; T. Sameshima et al., Effect of intestinalLactobacillus starter cultures on the behaviour of Staphylococcus aureusin fermented sausage, Int. J. Food Microbiol. 41(1):1-7 [1998]).

Optionally, after at least partial eradication of small intestinalbacterial overgrowth, use of antimicrobial agents or probiotic agentscan be continued to prevent further development or relapse of SIBO.

Another preferred method of at least partially eradicating smallintestinal bacterial overgrowth is by normalizing or increasing phaseIII interdigestive intestinal motility with any of several modalities toat least partially eradicate the bacterial overgrowth, for example, bysuitably modifying the subject's diet to increase small intestinalmotility to a normal level (e.g., by increasing dietary fiber), or byadministration of a chemical prokinetic agent to the subject, includingbile acid replacement therapy when this is indicated by low or otherwisedeficient bile acid production in the subject.

For purposes of the present invention, a prokinetic agent is anychemical that causes an increase in phase III interdigestive motility ofa human subject's intestinal tract. Increasing intestinal motility, forexample, by administration of a chemical prokinetic agent, preventsrelapse of the SIBO condition, which otherwise typically recurs withinabout two months, due to continuing intestinal dysmotility. Theprokinetic agent causes an in increase in phase III interdigestivemotility of the human subject's intestinal tract, thus preventing arecurrence of the bacterial overgrowth. Continued administration of aprokinetic agent to enhance a subject's phase III interdigestivemotility can extend for an indefinite period as needed to preventrelapse of the SIBO condition.

Preferably, the prokinetic agent is a known prokinetic peptide, such asmotilin, or functional analog thereof, such as a macrolide compound, forexample, erythromycin (50 mg/day to 2000 mg/day in divided doses orallyor I.V. in divided doses), or azithromycin (250-1000 mg/day orally).

However, a bile acid, or a bile salt derived therefrom, is anotherpreferred prokinetic agent for inducing or increasing phase IIIinterdigestive motility. (E. P. DiMagno, Regulation of interdigestivegastrointestinal motility and secretion, Digestion 58 Suppl. 1:53-55[1997]; V. B. Nieuwenhuijs et al, Disrupted bile flow affectsinterdigestive small bowel motility in rats, Surgery 122(3):600-08[1997]; P. M. Hellstrom et al., Role of bile in regulation of gutmotility, J. Intern. Med. 237(4):395-402 [1995]; V. Plourde et al.,Interdigestive intestinal motility in dogs with chronic exclusion ofbile from the digestive tract, Can. J. Physiol. Pharmacol.65(12):2493-96 [1987]). Useful bile acids include ursodeoxycholic acidand chenodeoxycholic acid; useful bile salts include sodium or potassiumsalts of ursodeoxycholate or chenodeoxycholate, or derivatives thereof.

A compound with cholinergic activity, such as cisapride (i.e.,Propulsid®; 1 to 20 mg, one to four times per day orally or I.V.), isalso preferred as a prokinetic agent for inducing or increasing phaseIII interdigestive motility. Cisapride is particularly effective inalleviating or improving hyperalgesia related to SIBO or associated withdisorders caused by SIBO, such as IBS, fibromyalgia, or Crohn's disease.

A dopamine antagonist, such as metoclopramide (1-10 mg four to six timesper day orally or I.V.), domperidone (10 mg, one to four times per dayorally), or bethanechol (5 mg/day to 50 mg every 3-4 hours orally; 5-10mg four times daily subcutaneously), is another preferred prokineticagent for inducing or increasing phase III interdigestive motility.Dopamine antagonists, such as domperidone, are particularly effective inalleviating or improving hyperalgesia related to SIBO or associated withdisorders caused by SIBO, such as IBS, fibromyalgia, or Crohn's disease.

Also preferred is a nitric oxide altering agent, such as nitroglycerin,,nomega-nitro-L-arginine methylester (L-NAME), N-monomethyl-L-arginine(L-NMMA), or a 5-hydroxytryptamine (HT or serotonin) receptorantagonist, such as ondansetron (2-4 mg up to every 4-8 hours I.V.;pediatric 0.1 mg/kg/day) or alosetron. The 5-HT receptor antagonists,such as ondansetron and alosetron, are particularly effective inimproving hyperalgesia related to SIBO, or associated with disorderscaused by SIBO, such as IBS, fibromyalgia, or Crohn's disease.

An antihistamine, such as promethazine (oral or I.V. 12.5 mg/day to 25mg every four hours orally or I.V.), meclizine (oral 50 mg/day to 100 mgfour times per day), or other antihistamines, except ranitidine(Zantac), famotidine, and nizatidine, are also preferred as prokineticagents for inducing or increasing phase III interdigestive motility.

Also preferred are neuroleptic agents, including prochlorperazine (2.5mg/day to 10 mg every three hours orally; 25 mg twice daily rectally; 5mg/day to 10 mg every three hours, not to exceed 240 mg/dayintramuscularly; 2.5 mg/day to 10 mg every four hours I.V.),chlorpromazine (0.25 mg/lb. up to every four hours [5-400 mg/day]orally; 0.5 mg/lb. up to every 6 hours rectally; intramuscular 0.25 /lb.every six hours, not to exceed 75 /mg/day), or haloperidol (oral 5-10mg/day orally; 0.5-10 mg/day I.V.). Also useful as a prokinetic agent,for purposes of the present invention, is a kappa agonist, such asfedotozine (1-30 mg/day), but not excluding other opiate agonists. Theopiate (opioid) agonists, such as fedotozine, are particularly effectivein alleviating or improving hyperalgesia related to SIBO or associatedwith disorders caused by SIBO, such as IBS, fibromyalgia, or Crohn'sdisease.

The preceding are merely illustrative of the suitable means by whichsmall intestinal bacterial overgrowth is at least partially eradicatedby treatment in accordance with the present method. These means can beused separately, or in combination, by the practitioner as suits theneeds of an individual human subject.

Optionally, treating further includes administering to the human subjectan anti-inflammatory cytokine or an agonist thereof, substantiallysimultaneously with or after at least partially eradicating thebacterial overgrowth of the small intestine, to accelerate or furtherimprove the symptom(s) of irritable bowel syndrome, fibromyalgia,chronic fatigue syndrome, depression, ADHD, or an autoimmune disease, orCrohn's disease. Useful anti-inflammatory cytokines include human IL-4,IL-10, IL-11, or TGF-β, derived from a human source or a transgenicnon-human source expressing a human gene. The anti-inflammatory cytokineis preferably injected or infused intravenously or subcutaneously.

Optionally, when the suspected diagnosis is irritable bowel syndrome,fibromyalgia, chronic fatigue syndrome, depression, ADHD, or anautoimmune disease, such as multiple sclerosis or systemic lupuserythematosus, symptoms are improved by administering an antagonist of apro-inflammatory cytokine or an antibody that specifically binds apro-inflammatory cytokine. The antagonist or antibody is administered tothe human subject substantially simultaneously with or after treatmentto at least partially eradicate the bacterial overgrowth. The antagonistor antibody is one that binds to a pro-inflammatory cytokine orantogonizes the activity or receptor binding of a pro-inflammatorycytokine. Pro-inflammatory cytokines include TNF-α, IL-1α, IL-1β, IL6,IL-8, IL-12, or LIF. The cytokine antagonist or antibody is preferablyderived from a human source or is a chimeric protein having a humanprotein constituent. The cytokine antagonist or antibody is preferablydelivered to the human subject by intravenous infusion.

Optionally, the method of treating irritable bowel syndrome,fibromyalgia, chronic fatigue syndrome, depression, attentiondeficit/hyperactivity disorder, an autoimmune disease, or Crohn'sdisease, further comprises administering an agent that modifies afferentneural feedback or sensory perception. This is particularly useful when,after at least partial eradication of SIBO, the subject experiencesresidual symptoms of hyperalgesia related to SIBO or associated with adisorder caused by SIBO, such as IBS, fibromyalgia, or Crohn's disease.Agents that modify afferent neural feedback or sensory perceptioninclude 5-HT receptor antagonists, such as ondansetron and alosetron;opiate agonists, such as fedotozine; peppermint oil; cisapride; adopamine antagonist, such as domperidone; an antidepressant agent; ananxiolytic agent; or a combination of any of these. Usefulantidepressant agents include tricyclic antidepressants, such asamitriptyline (Elavil); tetracyclic antidepressants, such asmaprotiline; serotonin re-uptake inhibitors, such as fluoxetine (Prozac)or sertraline (Zoloft); monoamine oxidase inhibitors, such asphenelzine; and miscellaneous antidepressants, such as trazodone,venlafaxine, mirtazapine, nefazodone, or bupropion (Wellbutrin).Typically, useful antidepressant agents are available in hydrochloride,sulfated, or other conjugated forms, and all of these conjugated formsare included among the useful antidepressant agents. Useful anxiolytic(anti-anxiety) agents include benzodiazepine compounds, such as Librium,Atavin, Xanax, Valium, Tranxene, and Serax, or other anxiolytic agentssuch as Paxil.

Representative methods of administering include giving, providing,feeding or force-feeding, dispensing, inserting, injecting, infusing,prescribing, furnishing, treating with, taking, swallowing, eating orapplying.

Eradication of the bacterial overgrowth is determined by detectionmethods described above, particularly in comparison with recordedresults from pre-treatment detection. After at least partiallyeradicating the bacterial overgrowth, in accordance with the presentmethod, the symptom(s) of irritable bowel syndrome, fibromyalgia,chronic fatigue syndrome, depression, ADHD, an autoimmune disease, orCrohn's disease are improved. Improvement in a symptom(s) is typicallydetermined by self-reporting by the human subject, for example by VASscoring or other questionnaire. Improvement in academic, professional,or social functioning, e.g., in cases of ADHD or depression can also bereported by others or can be observed by the clinician. Improvement(increase) in pain threshold, e.g., in subjects diagnosed withfibromyalgia, can be measured digitally, for example, by tender pointcount, or mechanically, for example, by dolorimetry. (F. Wolfe et al.,Aspects of Fibromyalgia in the General Population: Sex, Pain Threshold,and Fibromyalgia Symptoms, J. Rheumatol. 22:151-56 [1995]). Improvementin visceral hypersensitivity or hyperalgesia can be measured by balloondistension of the gut, for example, by using an electronic barostat. (B.D. Nabiloff et al., Evidence for two distinct perceptual alterations inirritable bowel syndrome, Gut 41:505-12 {1997]). Some improvement(s) insymptoms, for example systemic lupus erythematosus symptoms, such asrashes, photosensitivity, oral ulcers, arthritis, serositis, orimprovements in the condition of blood, kidney or nervous system, can bedetermined by clinical observation and measurement.

The present invention also relates to a kit for the diagnosis andtreatment of irritable bowel syndrome, fibromyalgia, chronic fatiguesyndrome, depression, ADHD, an autoimmune disease, or Crohn's disease.The kit is a ready assemblage of materials for facilitating thedetection and at least partial eradication of small intestinal bacterialovergrowth. The kit includes at least one, and most preferably multiple,air-tight breath sampling container(s), such as a bag, cylinder, orbottle, and at least one pre-measured amount of a substrate, such aslactulose (e.g., 10-20 g units) or glucose (e.g., 75-80 g units), formeasuring breath hydrogen and/or methane before and/or after a treatmentto at least partially eradicate SIBO. Alternatively, the kit containspre-measured amount(s) of isotope-labeled substrate as described above.The present kit also contains instructions for a user in how to use thekit to effectively corroborate a suspected diagnosis of irritable bowelsyndrome, fibromyalgia, chronic fatigue syndrome, depression, ADHD, anautoimmune disease, or Crohn's disease, in accordance with the presentdiagnostic method, and how to treat the underlying cause of any of theseconditions by at least partially eradicating SIBO.

Optionally, the kit also contains components useful for at leastpartially eradicating SIBO, for example, unitized amounts of anantimicrobial agent, such as neomycin, metronidazole, teicoplanin,doxycycline, tetracycline, ciprofloxacin, augmentin, cephalexin (e.g.,Keflex), penicillin, ampicillin, kanamycin, rifamycin, rifaximin, orvancomycin, or a 4- or 5-aminosalicylate compound, and/or a probioticagent, such as an inoculum of a species or strain of Bifidobacterium orLactobacillus, or a prokinetic agent, such as a peptide or functionalanalog thereof, macrolide compound, a bile acid, a bile salt, acholinergic compound, a dopamine antagonist, a nitric oxide alteringagent, a 5-HT receptor antagonist, a neuroleptic agent, a kappa agonist,or an antihistamine except ranitidine, famotidine, or nizatidine. Anycombination of these can be included in the kit. The kit optionallycontains an anti-inflammatory cytokine or an agonist thereof, or anantagonist or antibody effective against a pro-inflammatory cytokine.The kit optionally contains an agent that modifies afferent neuralfeedback or sensory perception, as described above, for alleviating orimproving hyperalgesia related to SIBO or associated with a disordercaused by SIBO, such as IBS, fibromyalgia, or Crohn's disease.

The components assembled in the kits of the present invention areprovided to the practitioner stored in any convenient and suitable waythat preserves their operability and utility. For example the componentscan be in dissolved, dehydrated, or lyophilized form; they can beprovided at room, refrigerated or frozen temperatures.

The foregoing descriptions for the methods and kits of the presentinvention are illustrative and by no means exhaustive. The inventionwill now be described in greater detail by reference to the followingnon-limiting examples.

EXAMPLES Example 1 Composition of the Database

Data were assembled from 202 human subjects from the Cedars-SinaiMedical Center GI Motility Program who completed an extensivequestionnaire of health history. These patients were all referred forlactulose breath hydrogen testing (LBHT) by more than 30 privategastroenterologists. These patients were selected by theirgastroenterologists to undergo breath testing, because they had symptomscompatible with SIBO. However, the questionnaire focused on general riskfactors, associated conditions, and symptoms found in these patients andnot specifically the incidence of SIBO. After antibiotic therapy, 59subjects actually returned for a follow up LBHT and a follow-upquestionnaire. This likely resulted in an underestimate ofresponsiveness to treatment, since only those who failed to respondadequately were likely to return to assess eradication of SIBO.

Example 2 Breath Hydrogen Testing

Subjects were tested after an overnight fast. At time zero, each subjectswallowed 15 ml of Chronulac formula, delivering 10 g of lactulose;every 5-20 min thereafter, for 2-4 hours, a 50 cm³ end-expiratory breathsample was taken with an airtight sampling bag. Each breath sample wasthen analyzed for hydrogen content with a gas chromatograph (QuintronModel DP, Quintron Instrument Co., Division of E. F. Brewer Co,Menomonee Falls, Wis. 53051), standardized using a QuinGas standard asinstructed by the manufacturer. Hydrogen peaks were plotted before andafter an antimicrobial treatment regimen for comparison. The normalrange for the second hydrogen peak was 0 to 20 ppm.

Example 3 Diagnosis and Treatment of Irritable Bowel Syndrome

The two hundred-two (202) human subjects were assessed for SIBO withLBHT. Of the 202 subjects in the database, 95 claimed to have been givena diagnosis of IBS. In addition, a symptom questionnaire was used todetermine whether these subjects fulfilled Rome criteria for IBS, andfour of the subjects failed to meet the Rome criteria. Crohn's diseasewas present in 14 of the subjects and four had a history of ulcerativecolitis. After these 22 subjects were excluded, 73 subjects remained.

Among the 107 subjects who stated that they had not previously beengiven a diagnosis of IBS, 78 met Rome criteria. After the 21 who hadCrohn's disease, five who had ulcerative colitis and one with shortbowel transit were excluded, 51 subjects remained. Data gathered fromthese subjects were pooled with data from the previous 73 subjects withsuspected IBS, yielding a total of 124 of the original 202 (61%)subjects with a suspected diagnosis of IBS.

Of the 124, 92 (74%) were positive for SIBO. However, of the 32 subjectsmeeting the Rome criteria, who were negative for SIBO, 14 had beentreated with antibiotics within 3 months prior to LBHT. Therefore, theincidence of SIBO among the 110 untreated subjects was 92 (84%), showinga strong association between a suspected diagnosis of IBS and thepresence of SIBO. After neomycin treatment (500 mg twice daily for tendays), 23 of these 92 returned for follow-up testing. On a visual analogscores (VAS), subjects were asked to rate their degree of post-treatmentimprovement. These 23 subjects reported a 60±31% improvement, although17 had only partial eradication of SIBO, based on their LBHT results.(FIG. 1).

There was a likely selection bias in the database due to the fact thatsubjects were referred for LBHT, because their physicians suspected theyhad SIBO. To correct for this bias, a pilot study was also conductedlooking at the incidence of bacterial overgrowth in patients with IBS.All patients between the ages of 18 and 65 referred to the Cedars-SinaiGI Motility Program who met Rome criteria for IBS, and who had had aprevious upper GI (small bowel) with follow-through (i.e., barium orGastrograffin imaging analysis) ruling out Crohn's disease andulcerative colitis, were asked to present to the GI motility laboratoryfor LBHT. Eight human subjects with a suspected diagnosis of IBS, basedon the Rome criteria, were tested for SIBO, using LBHT as described inExample 2. Seven of these patients (87.5%) were found to have SIBO basedon hydrogen peaks in a range of 80-250 ppm of hydrogen. Six of the 7subjects testing positive for SIBO returned approximately 10 days aftercompletion of a 10 day course of neomycin as described above. Neomycintreatment completely eradicated the SIBO in each of the six subjects,based on post-treatment breath hydrogen peaks in the normal range of0-20 ppm. The six subjects reported an average improvement in their IBSsymptoms of 65±28% (Range: 20-100%) on VAS scoring. FIG. 2 shows VAS forthe six subjects, based on a scale of 0-5, with 0 implying no pain and 5the most pain of life-time. It is clear from these results that at leastpartial eradication of bacterial overgrowth results in an improvement ingastrointestinal symptoms including bloating, gas, diarrhea, abdominalpain, sensation of incomplete evacuation and even constipation,associated with IBS. Additionally, significant extraintestinal symptomsof IBS, such as joint pain and fatigue, were also substantiallyimproved, and the degree of improvement was greater in subjects who hadcomplete eradication of SIBO.

Example 4 Diagnosis and Treatment of Fibromyalgia and Chronic FatigueSyndrome

Fibromyalgia: Of the 202 patients in the database, 37 (18%) had asuspected diagnosis of fibromyalgia. Of these 37, 28 tested positive forSIBO. However, of the nine who tested negative for SIBO, six had takenantibiotics within the preceding 3 months, and were excluded. Therefore,28 out of 30 (93%) of subjects with suspected fibromyalgia had SIBO,demonstrating a strong association between a suspected diagnosis offibromyalgia and the presence of SIBO.

After neomycin treatment (500 mg twice daily, 10-day course), ten ofthese 28 subjects returned, and post-treatment LBHT confirmed that SIBOhad been at least partially eradicated. These ten subjects reported a63±19% overall improvement in their symptoms by VAS scoring. FIG. 3compares the VAS scores for various symptoms reported by the subjectswith a suspected diagnosis of fibromyalgia before and after neomycintreatment. Symptoms included bloating, gas, diarrhea, joint pain andfatigue to treatment. Subjects were asked to identify the symptom mostimproved. Five subjects reported that pain was the most improved; threesubjects reported that the level of fatigue was most improved, and twoothers reported that their abdominal complaints improved the most. Therewas a negative correlation between the degree of improvement in the VASscoring and the amount of residual hydrogen peak seen in LBHT.(Pearson=−0.689, p=0.02; FIG. 4).

Subsequently, forty-six human subjects with FM (ACR criteria) entered adouble blind randomized placebo controlled trial. Each subject underwentLBHT, a tender point examination and completed a questionnaire at theinitial (baseline) and at every subsequent visit. Subjects wererandomized to receive neomycin (500 mg twice daily in liquid form) or amatched placebo, for 10 days. After completion of this treatment,subjects with persistent SIBO received antibiotics (open label) until atleast partially eradication was confirmed by LBHT. T-test was used tocompare the symptom scores of patients whose SIBO condition was at leastpartially eradicated with those whose SIBO was not at least partiallyeradicated.

Forty-two of the 46 FM patients (91.3%) were found to have SIBO. Six outof 20 patients (30%) in the neomycin group achieved complete at leastpartially eradication in the blinded arm. Only 6 subjects showed nodifference in the symptom score before and after the 10 d treatment.Twenty-eight subjects went on to open label treatment with 17 (60.7%)achieving complete at least partially eradication of SIBO. When symptomscores after at least partially eradication of SIBO on double blind oropen treatment were compared to baseline, there was significantimprovement in Tender Points, Tender Point Score, Hamilton DepressionScale, Fibromyalgia Impact Questionnaire (FIQ), Beck Depression Scale,Health Assessment Questionnaire (HAQ), VAS-Pain,VAS-Memory/Concentration and IBS-Quality of Life (QOL). (Initial data inTable 1). These results confirm that SIBO is associated withfibromyalgia, and that at least partially eradication of SIBO improvessymptoms in fibromyalgia.

TABLE 1 Selected Symptom Scores Double Blind Randomized PlaceboControlled Trial with Subjects Diagnosed with Fibromyalgia. SIBO SIBOnot eradicated eradicated eradicated vs. not (n = 25) (p = 15)eradicated Observation Baseline eradicated P-value Baseline eradicatedP-value P-value Tender Points 13.3 ± 2.9  10.3 ± 4.2  0.01 13.6 ± 2.0 12.1 ± 4.1  NS NS (TP) TP Score 20.3 ± 7.0  15.0 ± 9.1  0.01 23.7 ± 8.0 19.9 ± 9.7  NS NS FIQ 66.8 ± 18.2 49.5 ± 17.7 0.0001 72.7 ± 19.9 64.1 ±20.9 0.04 0.02 VAS-pain (mm) 80.7 ± 22.7 52.4 ± 28.5 0.00005 87.5 ± 19.676.2 ± 25.2 NS 0.01 HAQ 42.4 ± 10.5 37.7 ± 10.1 0.005 45.1 ± 11.2 43.9 ±12.1 NS NS

Chronic Fatigue Syndrome: Thirty of 202 subjects in the database (15.9%)had received a diagnosis of chronic fatigue syndrome. Of these 30subjects, 21 (70%) had SIBO as indicated by LBHT, but four out of thenine without SIBO had recently taken antibiotics. Therefore, theprevalence of SIBO was 21 out of 26 (81%) subjects with a diagnosis ofCFS. After treatment with neomycin (500 mg twice daily, 10-day course),nine of the 21 subjects diagnosed with CFS, returned for follow-up LBHTand questionnaire. LBHT showed that all nine subjects experienced atleast partially eradication of SIBO, and important symptoms of CFS weresubstantially improved after treatment. (Table 2).

TABLE 2 VAS scores by CFS patients reporting before and afteranti-biotic treatment. Symptom Before Antibiotic After AntibioticP-value Bloating 4.3 ± 1.0 2.3 ± 1.7 0.002 Fatigue 4.6 ± 1.0 3.5 ± 1.40.02 

Example 5 Autoimmune Diseases, Depression, and ADHD

SLE. Fifteen of the 202 (7.4%) subjects in the database had beendiagnosed with SLE. Of these 15 subjects, 13 (87%) had bacterialovergrowth, as indicated by LBHT. Four of the 15 subjects with SLEreturned for follow-up LBHT and questionnaire after treatment withneomycin (500 mg twice daily for 10 days). LBHT results for these fourwere negative for SIBO, and other significant symptoms weresignificantly improved after treatment. (Table 3).

TABLE 3 VAS scores by SLE patients reporting before and afteranti-biotic treatment. Symptom Before Antibiotic After AntibioticP-value Bloating 3.0 ± 2.0 1.3 ± 1.3 0.1 Joint Pains 2.5 ± 1.5 0.5 ± 0.6 0.04 Gas 3.3 ± 1.7 1.9 ± 1.7 0.3 Fatigue 4.6 ± 1.0 3.5 ± 1.4 0.3

Multiple Sclerosis: A 22-year-old female who presented with a history ofmultiple sclerosis symptoms and with plaques demonstrated on MRIimaging. A suspected diagnosis of multiple sclerosis had been made by aneurologist was based on various neuropathies of the peripheral nervoussystem, including numbness, tingling, and weakness in the lowerextremities, but this subject also had associated bloating, gas,distension and alteration in bowel habits. The subject also complainedof a significant fatigue and nausea. The subject underwent LBHT, whichdetected SIBO. She was subsequently treated with neomycin (500 mg twicedaily for 10 days), which at least partially eradicated the bacterialovergrowth. This was followed by complete resolution of her nausea,fatigue, bloating, gas distension and alteration in bowel habits. Inaddition, the subject showed dramatic improvement and resolution of herneuropathies. She no longer had numbness or tingling in the hands orfeet and was functioning quite well. Approximately 6-8 weeks after thisinitial response, the patient had a relapse of her symptoms, includingbloating, gas, distension and neuropathy. She had a repeat LBHT thatconfirmed a recurrence of SIBO. Upon re-treatment with neomycin (500 mgtwice daily for 10 days), she once again experienced complete resolutionof her symptoms.

Depression: A 73-year-old female presented with bloating, gas, abdominaldistention, and cramping for a period of 3 years prior to LBHT. Symptomsof depression first appeared concurrently with the first appearance ofbowel symptoms, and were serious enough that psychiatric hospitalizationhad been considered by her attending psychiatrist. The subject reportedfeeling very depressed and was convinced that life was not worth living.The subject's LBHT indicated the presence of a SIBO condition. Aftertreatment with neomycin (500 mg twice daily for 10 days), the subjectstated that she felt “100% better.” She reported that her depression wascompletely resolved and that her energy was back to normal. In addition,her bowel symptoms were also completely improved. The subject had beenprescribed eight different anti-depressant medications, all of whichwere discontinued as a result of her improvement.

ADHD: A 13 year-old female was brought in by her mother with a suspecteddiagnosis of attention deficit/hyperactivity disorder (AD type), made bya pediatrician. Concurrently, she also had significant bloating, gas andsome alteration in bowel habits. She had initially been referred fordiagnosis by her teachers and school counselors, because she had beenhaving difficulty performing in school for the previous two to threeyears, after having previously been a very good student. Prior to thedetection of SIBO, the subject had been treated with multiplepharmacologic agents for depression, including amitryptiline, with nonoticeable improvement in her symptoms.

The subject underwent LBHT that demonstrated the presence of SIBO. Thesubject was treated with neomycin (500 mg twice daily for 10 days) andafter complete at least partially eradication of the bacterialovergrowth, she had resolution of her bowel symptoms. Additionally, shestarted to get “A” averages in school again after being in the “C”range. She was able to concentrate better, and her teachers noticed adifference in her focus and attitude. Approximately two months later thesubject had a relapse in her attention problem which was concurrent witha recurrence of the bacterial overgrowth, as detected by LBHT. Afterrepeat treatment with neomycin (500 mg twice daily for 10 days), thesubject again responded with improved concentration and resolution ofbowel symptoms.

Example 6 Diagnosis and Treatment of Crohn's disease

Of the 202 subjects in the database, 39 (19%) had a suspected diagnosisof Crohn's disease. Of these 39, eight demonstrated short bowel transitand one subject produced neither hydrogen nor methane in LBHT; thesenine were excluded. Of the 30 remaining subjects, 22 had SIBO. However,of the eight subjects who had a negative LBHT result, five had beentreated with antibiotics within the preceding 3 months. If thesesubjects are excluded, 22 out of 25 (88%) subjects with a suspecteddiagnosis of Crohn's disease had SIBO, which shows a strong associationbetween a suspected diagnosis of Crohn's disease and the presence ofSIBO.

Of the 22 patients testing positive for the presence of SIBO, ninereturned after neomycin treatment (10-day course of 500 mg twice/daily)for LBHT, which showed at least partially eradication of SIBO. Thesenine patients reported a 57±32% (n=8 because one patient failed toreport percent improvement) overall improvement in their symptoms byVAS. If these subjects remained positive after antibiotic treatment withneomycin, metronidazole (Flagyl®),or ciprofloxacin, their improvementwas only 20±0% as opposed to 69±27% if the breath test was negative(p<0.05). FIG. 5 shows a dramatic improvement in the patients symptomsafter treatment. There was an especially notable reduction in bloodystools, diarrhea and fatigue.

As with the subjects with fibromyalgia, there was a negative correlationbetween the degree of improvement in the VAS scoring and the amount ofresidual hydrogen production (Pearson=−0.787, p=0.02; FIG. 6).

To correct for selection bias, a pilot study was conducted to determinethe incidence of SIBO in subjects who had received a suspected diagnosisof Crohn's disease at Cedars-Sinai Medical Center's IBD Center withinthe preceding three months. Six of these subjects underwent LBHT, ofwhom five (83%) were positive for SIBO.

Two of the six subjects returned for follow-up after antibiotic therapy(10-day course of neomycin). Post-treatment LBHTs showed that SIBO hadbeen completely at least partially eradicated in both subjects. Theyreported, respectively, a 60% and 80% overall improvement in theirsymptoms. This improvement was stated to include substantial reductionin diarrhea, gas and bloating.

Example 7 Response Stratification

There is a stratification in the degree of overgrowth and production ofhydrogen among the various diagnostic categories. For example, duringthe double blind study in the treatment of SIBO in fibromyalgia (Example4), it was noted that the level of hydrogen production during the LBHTwas much higher in this group of subjects as compared to those insubjects in the IBS incidence study described in Example 3. Given thatthe bacterial load is related to the level of hydrogen production, thisimplies that the degree of overgrowth is higher in patients withfibromyalgia compared to subjects with IBS.

The stratification of breath hydrogen levels with respect to diagnosticcategories is as follows: IBS/Crohn's Disease (40-70 ppm of hydrogen);CFS (50-100 ppm of hydrogen); and FM (100-250 ppm of hydrogen).

Example 8 Intestinal Dysmotility Associated with IBS and FM

Clinical experience showed that SIBO tends to recur after anti-biotictreatment within about 2 months. To demonstrate that a lack of phase IIIinterdigestive motility is responsible for SIBO in subjects with IBS orfibromyalgia, antreduodenal manometry was conducted in human subjectsdiagnosed with IBS or FM.

Antreduodenal Manometry. PhaseIII interdigestive (fasting) motility wasassessed in 15 human subjects. An antreduodenal manometry was performedby placing an 8-channel small bowel manometry catheter (each channelspaced 5 cm apart) into the small bowel using fluoroscopic guidance.After placement of the catheter, manometric recordings were made with anArndorffer perfusion system with signals collected usingMedtronics/Synectics Polygraf and associated Polygram software. Datawere assessed for the characteristics of interdigestive motility.

IBS. Phase III interdigestive motility was assessed for a six-hourperiod in 15 human subjects having a suspected diagnosis of IBS, asdefined by Rome Criteria, corroborated by concomitant SIBO. Of these 15subjects, 13 (86%) had no detectable phase III interdigestive motilityduring the period of study. One subject (7%) had phase IIIinterdigestive motility of short duration (<3 minutes), and one subject(7%) had normal phase III interdigestive motility.

Fibromyalgia. Phase III interdigestive motility was assessed in sevenhuman subjects having a suspected diagnosis of fibromyalgia corroboratedby the presence of SIBO. Of these seven subjects, six (86%) lackeddetectable phase HI interdigestive motility, and one subject (14%) hadmotility of less than normal peristaltic amplitude. The duration ofstudy in the patients with fibromyalgia averaged 216±45 minutes in thefasting state.

Example 9 Treatment of IBS with a Prokinetic Agent

Erythromycin, as a motilin agonist, can induce phase III ofinterdigestive motility. (E.g., M. J. Clark et al., Erythromycinderivatives ABT229 and GM 611 act on motilin receptors in the rabbitduodenum, Clin. Exp. Pharmacol. Physiol. 26(3):242-45 [1999]).Therefore, two subjects with recurrent IBS symptoms received prokinetictreatment with erythromycin.

The two subjects were a 55-year-old female and a 43-year-old female,both diagnosed with IBS. SIBO was detected in these subjects by LBHT.Antibiotic treatment of the SIBO resulted in greater than 90%improvement in symptoms. However, IBS symptoms recurred three to fourweeks later, concurrent with a return of the SIBO condition. Subsequentcourses of antibiotic treatment resulted in a similar pattern ofimprovement followed by a rapid recurrence of IBS symptoms in bothsubjects. Antreduodenal manometry was performed, demonstrating a lack ofphase III of interdigestive motility, and erythromycin (50 mg daily) wasprescribed to the subjects. The two subjects subsequently remained freeof IBS symptoms and SIBO for at least 18 months and six months,respectively.

These results demonstrate the effectiveness of prokinetic treatment witherythromycin in preventing the recurrence of SIBO and IBS symptoms insubjects diagnosed with IBS.

Example 10 Treatment of SIBO-Related Hyperalgesia

An adult male subject with a suspected diagnosis of IBS was found tohave SIBO, as detected by LBHT. Anorectal manometry revealed rectalhypersensitivity in this subject. After eradication of his SIBOcondition with antibiotic treatment, a repeat anorectal manometry showedthat his rectal hyperalgesia had resolved.

Two adult female subjects with IBS required additional pharmacologicmanipulations to treat their SIBO-related hyperalgesia In the firstcase, SIBO was eradicated by antibiotic treatment. However, the subjectcomplained of persistent feelings of rectal distension, consistent withresidual hyperalgesia related to SIBO. The subjected was thenadministered Colpermin (peppermint oil) capsules and Elavil (5 mg takenat night) that alleviated her SIBO-related hyperalgesic symptoms,presumably by reducing intestinal wall tension and decreasingmechanoreceptor activation.

The second female subject with a diagnosis of IBS was also found to haveSIBO, as detected by LBHT. Her SIBO was eradicated by a combinedtreatment with antibiotic, intestinal lavage with Go-Lytely, andcisapride (10 mg tid) to increase her abnormally low phase IIIinterdigestive motility. After eradication of SIBO, this subjectsimilarly complained of persistent SIBO-related hyperalgesic symptoms ofthe bowel. Administration of Colpermin (peppermint oil) thensuccessfully alleviated the hyperalgesia, presumably by reducing themechanoreceptor feedback for rectal distension.

The foregoing examples being illustrative but not an exhaustivedescription of the embodiments of the present invention, the followingclaims are presented.

We claim:
 1. A method of diagnosing irritable bowel syndrome comprising:detecting the presence of small intestinal bacterial overgrowth in ahuman subject having at least one symptom associated with a suspecteddiagnosis of irritable bowel syndrome, whereby the suspected diagnosisof irritable bowel syndrome is corroborated by the presence of smallintestinal bacterial overgrowth.
 2. The method of claim 1, whereindetecting the presence of a bacterial overgrowth is by intestinalsampling from said human subject.
 3. The method of claim 2, whereinsampling is of cellular, fluid, fecal, or gaseous matter contained bythe intestinal lumen or comprising part of the lumenal wall.
 4. A methodof diagnosing irritable bowel syndrome, comprising: detecting byanalyzing the content of a gas mixture, the presence of small intestinalbacterial overgrowth in a human subject having at least one symptomassociated with a suspected diagnosis of irritable bowel syndrome, saidgas mixture being at least partially produced by the intestinalmicroflora of said human subject and being exhaled by said human subjectafter ingesting a controlled quantity of a substrate, whereby thesuspected diagnosis of irritable bowel syndrome is corroborated by thepresence of small intestinal bacterial overgrowth.
 5. The method ofclaim 4, wherein the substrate is an isotope-labeled sugar or a sugarthat is incompletely digested by a human.
 6. The method of claim 5,wherein the sugar is glucose, lactose, lactulose or xylose.
 7. Themethod of claim 4, wherein the content of methane, carbon dioxide, orhydrogen gas in the exhaled gas mixture is analyzed.
 8. The method ofclaim 4, wherein analyzing the exhaled gas mixture is by gaschromatography.
 9. The method of claim 5, wherein the sugar is anisotope-labeled sugar; and analyzing the exhaled gas mixture is by massspectrometry or radiation detection.
 10. The method of claim 9, whereinthe content of methane, hydrogen or carbon dioxide in the exhaled gasmixture is analyzed.